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ORIGINAL RESEARCHpublished: 21 October 2016

doi: 10.3389/fpls.2016.01564

Edited by:Janin Riedelsberger,

University of Talca, Chile

Reviewed by:Martin Hajduch,

Slovak Academy of Sciences,Slovakia

Andrea Ariani,University of California, Davis, USA

*Correspondence:Henry T. Nguyen

nguyenhenry@missouri.edu

Specialty section:This article was submitted to

Plant Physiology,a section of the journal

Frontiers in Plant Science

Received: 29 July 2016Accepted: 04 October 2016Published: 21 October 2016

Citation:Song L, Nguyen N, Deshmukh RK,

Patil GB, Prince SJ, Valliyodan B,Mutava R, Pike SM, Gassmann W

and Nguyen HT (2016) Soybean TIPGene Family Analysis and

Characterization of GmTIP1;5 andGmTIP2;5 Water Transport Activity.

Front. Plant Sci. 7:1564.doi: 10.3389/fpls.2016.01564

Soybean TIP Gene Family Analysisand Characterization of GmTIP1;5and GmTIP2;5 Water TransportActivityLi Song1, Na Nguyen1, Rupesh K. Deshmukh2, Gunvant B. Patil1, Silvas J. Prince1,Babu Valliyodan1, Raymond Mutava1, Sharon M. Pike3, Walter Gassmann3 andHenry T. Nguyen1*

1 Division of Plant Science, National Center for Soybean Biotechnology, University of Missouri, Columbia, MO, USA,2 Departement de Phytologie, Laval University, Quebec, QC, Canada, 3 Division of Plant Sciences and Interdisciplinary PlantGroup, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, USA

Soybean, one of the most important crops worldwide, is severely affected by abioticstress. Drought and flooding are the major abiotic stresses impacting soybean yield.In this regard, understanding water uptake by plants, its utilization and transport hasgreat importance. In plants, water transport is mainly governed by channel formingaquaporin proteins (AQPs). Tonoplast intrinsic proteins (TIPs) belong to the plant-specific AQP subfamily and are known to have a role in abiotic stress tolerance. Inthis study, 23 soybean TIP genes were identified based on the latest soybean genomeannotation. TIPs were characterized based on conserved structural features andphylogenetic distribution. Expression analysis of soybean TIP genes in various tissuesand under abiotic stress conditions demonstrated tissue/stress-response specificdifferential expression. The natural variations for TIP genes were analyzed using wholegenome re-sequencing data available for a set of 106 diverse soybean genotypesincluding wild types, landraces and elite lines. Results revealed 81 single-nucleotidepolymorphisms (SNPs) and several large insertions/deletions in the coding region ofTIPs. Among these, non-synonymous SNPs are most likely to have a greater impact onprotein function and are candidates for molecular studies as well as for the developmentof functional markers to assist breeding. The solute transport function of two TIPs wasfurther validated by expression in Xenopus laevis oocytes. GmTIP1;5 was shown tofacilitate the rapid movement of water across the oocyte membrane, while GmTIP2;5facilitated the movement of water and boric acid. The present study provides an initialinsight into the possible roles of soybean TIP genes under abiotic stress conditions. Ourresults will facilitate elucidation of their precise functions during abiotic stress responsesand plant development, and will provide potential breeding targets for modifying watermovement in soybean.

Keywords: aquaporin, tonoplast intrinsic proteins (TIPs), water transporter, soybean, abiotic stress, expression,SNP

INTRODUCTION

The need for sustainable production of food is a critical issue for human and environmentalhealth due to the continuously growing global population. Soybean, being a source of edibleoil and protein rich meal, is considered a promising crop to fulfill the increasing food demand(Krishnamurthy and Shivashankar, 1975). Soybean seed contains over 40% protein and 20%

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oil. However, stresses imposed by environmental factors greatlyaffect soybean yield and quality. Water is one of the importantfactors causing severe yield losses either with excess availabilityfrom flooding or with limitation resulting from drought. Plantscombat such stresses by regulating water distribution at differentlevels, such as the vascular system and the permeability of plasmamembranes. Aquaporins (AQPs), a class of channel formingproteins, facilitate transport of water and many other solutesacross cellular membranes (Javot and Maurel, 2002; Maurelet al., 2002; Tyerman et al., 2002). AQPs are integral membraneproteins that belong to the major intrinsic protein (MIP) family.In higher plants, MIPs are classified into five subfamilies,including plasma membrane intrinsic proteins (PIPs), tonoplastintrinsic proteins (TIPs), NOD 26-like intrinsic proteins (NIPs),small basic intrinsic proteins (SIPs), and uncategorized intrinsicproteins (XIPs) (Chaumont et al., 2001; Kaldenhoff and Fischer,2006; Danielson and Johanson, 2008).

It has been shown that the expression of TIP genes variesin different tissue, hormone and abiotic treatments. ZmTIP2-3 transcripts was detected only in maize root tissues and wasinduced by salt and water stresses (Lopez et al., 2004). CottonGhTIP1:1 transcripts mainly accumulated in roots and hypocotylsunder normal conditions, but were dramatically up-regulated incotyledons and down-regulated in roots within a few hours aftercotton seedlings were cold-treated (Li et al., 2009). Several root-specific RB7-type TIP genes have been identified fromArabidopsisthaliana (Yamamoto et al., 1990), Solanum tuberosum (Heinrichet al., 1996), Petroselinum crispum (Roussel et al., 1997),Helianthus annuus (Sarda et al., 1999), Mesembryanthemumcrystallinum (Kirch et al., 2000). Recently, one strawberry RB7-type TIP gene, FaRB7 also exhibited a root-specific expressionpattern (Vaughan et al., 2006).

Tonoplast intrinsic proteins genes also have been reported tobe involved in the elevation of abiotic stress tolerance in severalplant species. Notably, the TIP gene TsTIP1;2 from Thellungiellasalsuginea provided increased tolerance against drought, saltand oxidative stresses when ectopically expressed in Arabidopsis(Wang et al., 2014). Similarly, increased salinity tolerance wasachieved with the heterologous expression of tomato SITIP2;2in Arabidopsis (Xin et al., 2014). In soybean, Zhang et al.(2016) observed that the expression pattern of GmTIP2;3 wasaffected by PEG and ABA, and overexpressing GmTIP2;3 in yeastcells improved osmotic stress tolerance. However, another TIP2gene, GsTIP2;1 cloned from Glycine soja, resulted in reducedtolerance to salt and dehydration stress when overexpressedin Arabidopsis (Wang et al., 2011). Such contrasting resultsindicate diverse regulation of TIPs within the subfamily that maybe due to tissue-specific expression patterns, since GmTIP2;3was found to be highly expressed in roots whereas GsTIP2;1showed comparatively higher expression in leaves. Higherwater movement by GmTIP2;3 in roots can be correlatedwith efficient water uptake, leading to enhanced osmotic stresstolerance. In contrast, water movement regulated by GsTIP2;1seems to increase water loss through transpiration. Similarobservations have been reported in rice, where unbalancedexpression of most aquaporins in leaves compared to rootis thought to result in rapid depletion of leaf water and

subsequent inhibition of photosynthesis (Nada and Abogadallah,2014).

Slow wilting is an important physiological parameter to studywater stress tolerance in soybean. Genetic variation observed forthe slow wilting trait has proven very useful for improving yieldunder drought conditions (Sloane et al., 1990). Several genotypesdisplay heritable variation for the slow wilting phenotype. Forinstance, Sloane et al. (1990) observed delayed wilting in PI416937 and PI 471938 soybean lines under drought conditionsin the field. Recently, two new genotypes, PI 567731 and PI567690, have been identified for the slow wilting trait under fieldconditions (Pathan et al., 2014). Interestingly, a study conductedwith slow wilting soybean line PI416937 has revealed associationbetween AQPs and hydraulic conductance (Sadok and Sinclair,2010a,b, 2012). Devi et al. (2015) reported down-regulation ofAQPs under high vapor pressure deficit (VPD) conditions inPI 416937, which may be due to the reduced uptake of water,resulting in conservation for later availability. Recently, ourstudy has identified several differentially expressed AQP genesamong the slow wilting and fast wilting soybean lines (Princeet al., 2015). These results prompted us to undertake detailedstudies of candidate AQPs thought to be involved in soybeanabiotic stress tolerance. (Verkman et al., 2014; Madeira et al.,2016).

In the present study, a comprehensive analysis of thesoybean TIP gene family was carried out including phylogeneticrelationships, chromosomal location, gene duplication status,gene structure, conserved motif, expression profiling underabiotic stress, and natural variation in soybean wild types,landraces and elite lines. The water transport function of twoGmTIP proteins was validated through oocyte experiments.Xenopus laevis oocytes have very low background activity thathelps to achieve a high signal-to-noise ratio as required to studytransporters. Therefore, Xenopus oocytes have been routinelyused for the evaluation of solute permeability by several differenttransporters (Osawa et al., 2006; Verkman et al., 2014). Moreparticularly, AQPs are frequently analyzed using X. laevis oocytes(Maurel et al., 1993; Deshmukh et al., 2015). These data willcontribute to future studies to functionally characterize TIPproteins in soybean.

MATERIALS AND METHODS

Identification and StructuralOrganization of Tonoplast IntrinsicProtein (TIP) GenesThe Arabidopsis TIP2;1 amino acid sequence was used as queryto perform a database search using BLASTP against predictedproteins in the G. max Wm82.a2.v1 genome derived fromPhytozome databases. Sequence with at least 50% identity withthe query sequence was classified as candidate GmTIPs. BLASThits with less than a 100 bitscore were removed. Manualcuration was then performed to match TIPs identified in thepresent study with those reported earlier by Deshmukh et al.(2013). The genomic sequences, CDS, and protein sequences

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for all GmTIPs were retrieved from Phytozome (V111). NovelTIP genes identified with the recent version of the soybeangenome annotation were characterized for the aromatic/arginine(Ar/R) selectivity filters (SFs), Froger’s residues, Asn-Pro-Ala(NPA) motifs and the spacing between NPAs. The exon/intronorganizations of GmTIPs was visualized with the Gene StructureDisplay Server program (Hu et al., 2015; GSDS2).

Identification of Conserved Protein Motifand Channel Structure PredictionThe protein sequences were analyzed to identify conservedprotein motifs (Motif scan) using the ‘Multiple EM forMotif Elicitation’ (MEME) program (Bailey et al., 2006).Transmembrane domains in newly identified TIPs were predictedusing TOPCONS software tools3. Protein structures weremodeled based on the structure of Arabidopsis TIP2;14. The PoreWalker tool was used to predict the pore feature5 (Pellegrini-Calace et al., 2009).

Phylogenetic Tree AnalysisMultiple sequence alignments were conducted with the aminoacid sequence of GmTIPs. Subsequently, a phylogenetic tree wasconstructed using the Maximum-Likelihood method providedin the MEGA 6.0 software tool (Tamura et al., 2013). Thereliability of an inferred tree was confirmed with bootstrapanalysis performed with 1,000 replications.

Chromosomal Distribution and GeneDuplications in GmTIPsThe chromosomal location of all GmTIP genes was obtainedthrough BLASTN searches against the G. Max Wm82.a2.v1genome database in Phytozome. GmTIPs were located onsoybean chromosomes based on physical positions. To furtheranalyze gene duplication events, synonymous substitution (Ks)and non-synonymous substitution (Ka) rates were downloadedfrom the Plant Genome Duplication Database (PGDD) database6.The date of duplication events was subsequently estimatedaccording to the equation T = Ks/2λ, in which the meansynonymous substitution rate (λ) for soybean is 6.1 × 10−9

(Lynch and Conery, 2000).

Expression Profiling Using RNA-seqDatasetsThe RNA-seq data generated by Libault et al. (2010) for ninedifferent tissues including flower, leaves, nodules, pod, root,root hair, seed, shoot apical meristem, and stem were used toanalyze expression patterns of GmTIPs. Expression profiling ofGmTIPs in leaf tissues of PI 567690 (drought tolerant) and Pana(drought susceptible) grown under drought conditions, and PI

1https://phytozome.jgi.doe.gov2http://gsds.cbi.pku.edu.cn/3http://topcons.cbr.su.se/4http://swissmodel.expasy.org/interactive5http://www.ebi.ac.uk/thornton-srv/software/PoreWalker/6http://chibba.agtec.uga.edu/

408105A (flooding tolerant) and S99-2281 (PI 654356, floodingsusceptible) grown under flooding conditions, was extractedfrom earlier reported data (Mutava et al., 2015; Prince et al., 2015;Syed et al., 2015). Briefly, at the V5 stage (five unfolded trifoliateleaves), drought stress was imposed by withdrawing water for21 days and flooding stress was imposed by overwatering for15 days. Similarly, RNA-seq data for Williams 82 plants subjectedto very mild stress (VMS), mild stress (MS), and severe stress (SS)conditions, as well as recovery from severe stress after re-watering(SR), was used to study expression of GmTIPs (Song et al., 2016).Hierarchical clustering of expression data was performed usingdCHIP software (Li and Wong, 2001).

Analysis of Synonymous andNon-synonymous SNP Variants in 106Soybean LinesAll single-nucleotide polymorphism (SNP) datasets located inexonic regions were extracted from whole genome re-sequencing(sequencing depth approximately 15X) data (Valliyodan et al.,2016) as described by Patil et al. (2016). Annotation and effectprediction for the SNPs and other variants were performed usingSnpEff7. SNPs were further classified into synonymous and non-synonymous categories.

Xenopus laevis Oocyte AssayFull length cDNAs of soybean aquaporins were cloned intothe Xenopus expression vector pOO2, which contains 5′ and3′ UTR regions of the X. laevis β-globin gene, includingan extended polyA tract for improved mRNA stability andexpression (Ludewig et al., 2002). cRNA was synthesized usingthe SP6 Ambion mMessage mMachine kit with linearized pOO2constructs as templates. Oocytes were isolated and maintained asdescribed (Pike et al., 2009) and injected with 46 ng of cRNAper oocyte. Water transport was evaluated by perfusing oocyteswith hypoosmotic ND96 solution and monitoring swelling overtime (Maurel et al., 1993; Durbak et al., 2014). In addition,boron transport was evaluated by replacing the NaCl-containingND96 with an isoosmotic boron-ND96 solution and monitoringswelling over time (Durbak et al., 2014).

RESULTS

Genome-Wide Identification of TonoplastIntrinsic Proteins in SoybeanA total of twenty-three TIP genes was identified based on acomprehensive phylogenetic tree analysis within the GmMIPgene family (Deshmukh et al., 2013; Zhang et al., 2013). Here TIPgenes were reanalyzed based on a recently released version of thesoybean genome annotation (Wm82.a2.v1). The number of TIPsidentified in soybean is in good agreement with earlier studiesperformed with a previous genome annotation (Deshmukh et al.,2013; Zhang et al., 2013). However, the transcript of GmTIP4;2(Glyma04G08830) reported in earlier studies seems to have

7http://snpeff.sourceforge.net

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been based on a mispredicted gene model and was thereforeremoved from the Wm82.a2.v1 annotation. Another TIP gene,Glyma12g01490, earlier reported as a pseudo-gene because oftranscript truncation, was determined to generate a full-lengthtranscript (new ID Glyma.12G012300) in the new version. Twoother TIP pseudogenes reported in earlier studies were notpresent in the new genome version. Therefore, one gene wasremoved and one added, resulting in a corrected set of 23TIPs. The gene names, gene IDs and locations in the genomeare listed in Supplementary Table S1. The newly identified TIPgene (Glyma12G01490) was named GmTIP5;2 according to theclustering in the phylogenetic tree.

Phylogenetic Relationship and GeneStructureTo get a better understanding of the evolutionary history, anunrooted phylogenetic tree was constructed using the Maximum-Likelihood (ML) method on the basis of multiple sequencealignment of the 23 soybean TIP proteins (Figure 1A). Accordingto the ML phylogenetic tree, the TIP family is divided intofive subgroups designated as Group 1–Group 5. Group 1, thelargest clade, contains nine members, representing 39.1% ofthe total TIP genes. Group 4 constitutes the smallest cladewith only one member. To gain further insights into theevolutionary relationships among GmTIP genes, the exon/intronstructures of individual GmTIP genes were predicted based onthe alignment of CDS sequences with corresponding genomicDNA sequences. As illustrated in Figure 1B, 20 out of 23 GmTIPgenes have three exons, while the remaining three only possesstwo exons. Genes within the same clade demonstrated similarexon/intron distribution patterns in terms of exon/intron length,with the exception of GmTIP5;2 containing a short secondexon.

Chromosomal Location and Duplicationof Soybean TIP GenesThe 23 TIP genes were unevenly distributed on 16 of the20 soybean chromosomes (Figure 2), with three GmTIPs onchromosome 13, and two GmTIPs each on chromosomes 9, 10,11, 12, and 19. The remaining chromosomes only contained oneTIP each.

During evolution, the soybean genome has undergone tworounds of whole genome duplication (Schmutz et al., 2010).In order to examine the duplication patterns of soybeanTIP genes, the PGDD was searched to identify segmentallyduplicated pairs, and tandem duplication was identified basedon the gene loci. No tandem duplication was found in thisgene subfamily. To identify duplicated pairs, synonymous (Ks)and non-synonymous substitution (Ka) distance values werecalculated and the Ka/Ks ratios were used to evaluate theduplication time. The Ka/Ks ratio for each segmentally duplicatedgene pair varied from 0.06 to 0.28 (Supplementary Table S2). Thisanalysis suggests that all mutations in paralogous GmTIP genesare neutral or disadvantageous, as their Ka/Ks ratios were lessthan 1. We found that the five closest branches of soybean TIPgenes experienced duplication during the soybean whole genome

duplication period, while the others were duplicated 67 Mya orearlier. The duplicated GmTIPs exist in the form of sister pairs inthe phylogenetic tree (Figure 1) and are shown linked by dottedlines in Figure 2.

Sequence and Conserved DomainAnalysis in GmTIP Gene FamilyThe protein size of TIP members varied from 238 to 256amino acids with 78–99% identity in each subgroup. All soybeanGmTIP proteins contain two NPA motifs, and the amino acidsrepresenting the Ar/R selectivity filter (SF) and Froger’s residuesare highly conserved. The Ar/R SF in the newly identifiedGmTIP5;2 consists of the amino acid sequence S-V-G-C, and thespacing between the NPA domains is 110 amino acids, whichis consistent with other members of the GmTIP5 subfamily(Supplementary Table S3). Similarly, the sequence A-A-Y-W is acommon feature for both GmTIP5;1 and GmTIP5;2.

Tertiary protein structure predicted for TIPs showed sixconserved transmembrane domains. However, the threedimensional geometry of the pore structure obtained withPoreWalker software (Pellegrini-Calace et al., 2009) showedsubstantial variation in pore size and constrictions in the pore.The tertiary structure and pore morphology for GmTIP1;5 andGmTIP2;5 are shown in Figure 3.

The characterization of these aquaporins as TIP genes wasbased on predicted amino acid similarities with known TIPsfrom other plant species, with location in the tonoplast ofthe corresponding proteins needing experimental verification.However, the predicted localization of members of the GmTIPsubfamily was very diverse, including cytosol, plasma membrane,endoplasmic reticulum, vacuole, mitochondria, and chloroplast(Zhang et al., 2013). We further searched for conserved motifsin GmTIP proteins with the MEME program to gain additionalinsights into their diversity. As shown in Figure 4, 10 conservedmotifs designated as motif 1 to motif 10 were found. All GmTIPspossess motifs 1, 2, and 5. Most of the GmTIPs contain motifs1 to 8, whereas motif 9 and motif 10 were exclusively presentin the GmTIP5 subgroup. Motif 3 was not present in GmTIP1;1,GmTIP4;1, and GmTIP5;2. Motif 4 was not present in GmTIP1;4,GmTIP5;1, and GmTIP5;2.

Differential Expression of Soybean TIPsin Soybean Tissues and Under AbioticStress ConditionsVery diverse expression patterns for GmTIPs were observedin the transcriptome data representing nine different tissues,namely flower, leaf, nodule, pod, root, root hair, seed, shootapical meristem, and stem tissue (Libault et al., 2010). Mostof the GmTIPs showed tissue specific expression (Figure 5).For example, GmTIP1;1, 1;2, 1;3, 1;4, 2;6, 2;7, and 4;1were highly expressed in root, but have relatively lowerexpression levels in other tissues. Similarly, GmTIP1;5, 1;6,2;4, and 2;5 were highly expressed in stem, but have arelatively lower expression level in other tissues. No geneshowed higher expression levels in leaves and root hairs,while most phylogenetically paired genes showed similar

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FIGURE 1 | Phylogenetic relationship and exon-intron structure of soybean Tonoplast intrinsic proteins (TIPs). (A) The unrooted tree was constructed viaalignment of full-length amino acid sequences from soybean using MEGA6 software by the neighbor-joining method. (B) Lengths of the exons and introns of eachTIP gene are displayed proportionally. Exons and introns are indicated by yellow rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicatedby blue rectangles.

expression patterns. The GmTIP3 subgroup was only expressedin seed tissue, although this pattern broadened under stressconditions (see below). Interestingly, two phylogenetic gene pairsdisplayed different expression patterns (GmTIP2;1/GmTIP2;2,GmTIP1;8/GmTIP1;9), indicating neofunctionalization.

The tissue specific expression pattern of GmTIPs wasinvestigated in the Williams 82 genotype under varying water-deficit conditions (Figure 6A). GmTIP3;2, 3;3, and 3;4 wereup-regulated in shoots under serious drought condition, evenafter water-recovery, and down-regulated in all other tissuesand conditions. Interestingly, the whole GmTIP2 subfamily wasdown-regulated under very mild drought stress in leaf tissueand up-regulated after severe drought stress and water-recovery,

except one (GmTIP2;2). These results indicated that a genesubfamily may share similar regulatory elements, and alsohave similar physiological functions. Most of the root-specificexpressed genes were induced under varying water-deficitconditions.

To further investigate the expression profiles of TIP genesunder drought and flooding conditions, the expression patternsof TIP gene families were extracted from two RNA-seq datasets(Prince et al., 2015 and unpublished data) (Figure 6B). PI 567690is a slow wilting soybean line, and Pana is a fast wilting lineunder drought conditions (Pathan et al., 2014; Mutava et al.,2015). GmTIP2;1, 2;2, 2;3, 2;4, 2;5, 3;2, and 5;1 mRNA levelswere induced in leaves of Pana, but decreased in leaves of PI

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FIGURE 2 | Chromosomal locations and gene duplication events of soybean TIP gene family members. The segmentally duplicated gene pairs are linkedby black dotted lines. The left scale represents physical distance along the chromosomes in megabases (Mb). Chromosome numbers are shown at the top of eachvertical gray bar.

567690 after drought stress. PI 408105A is a flooding tolerantline, and S99-2281 is a flooding sensitive line (Mutava et al.,2015). GmTIP1;2, 1;3, 1;4, 1;5, 1;6, 1;9, 2;6, and 4;1 mRNA levelswere induced in leaves of S99-2281, but decreased or were notinduced in leaves of PI 408105A after flooding stress. Conversely,GmTIP1;8 and 2;7 were induced in PI 408105A and decreased inS99-2281. These results indicated that GmTIP genes could play arole under drought or flooding stress.

The expression pattern of the newly identifiedGmTIP5;2 couldnot be determined because all reads from the above RNA-seqdatasets were aligned to the previous version of the G. maxreference genome, Gmax1.1, and Phytozome v9.0. GmTIP3;1 wasnot induced or decreased in any tissues (Williams 82) or PI linesunder abiotic stress conditions (Figures 6A,B). Also GmTIP3;1,GmTIP3;4, GmTIP3;3, and GmTIP1;7, did not show any responseto abiotic stress in the PI or cultivar lines (Figure 6B).

Analysis of SNP Variation of the GmTIPGene Family in 106 Soybean LinesSingle-nucleotide polymorphisms located in the coding regionsof GmTIPs were identified to investigate the genetic variationwithin this gene family in diverse soybean lines (Supplementary

Table S4). A total of 81 SNPs were observed in the 23 GmTIPgenes. These SNPs exhibited an uneven distribution: the GmTIP5subfamily contains the highest number of SNPs compared toother subfamilies, whereas no SNPs were found in GmTIP2;1and GmTIP2;3. Out of 81 SNPs, one SNP located in GmTIP5;1generates a premature stop codon. Thirty-six SNPs were non-synonymous and were distributed in the coding regions of 14GmTIPs. Some deletions were found in non-synonymous SNPlocations, which cause protein-coding changes. Not a single non-synonymous SNP was identified within the remaining sevengenes (GmTIP1;2, GmTIP1;5, GmTIP1;9, GmTIP2;4, GmTIP2;5,GmTIP2;6, and GmTIP4;1). SNPs unique to G. soja lines (PI#highlighted red) are summarized in Supplementary Table S4.Only 18 unique SNPs were identified in seven G. soja lines,and eight out of these 18 SNPs were non-synonymous. Thepattern of SNP distributions correlates well with the phylogeneticdistribution of TIPs (Figure 1; Supplementary Table S2). Thissequence information enabled us to identify novel GmTIPalleles from soybean lines that will serve as valuable breedingresources.

Genetic variations were further investigated in four soybeanslow wilting lines (PI 471938, PI 416937, PI 567690, and PI567731) to establish associations between SNPs and the slow

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FIGURE 3 | Protein tertiary structure showing pore morphology of GmTIP1;5 and GmTIP2;5. (A) Tertiary structures comprised of six transmembranedomains and water molecules (red) passing through the pores of GmTIP1;5 (left) and GmTIP2;5 (right) visualized with CLC genomic workbench. (B) Cross sectionsof the proteins showing pore. (C) Pore diameter profile of GmTIP1;5 (left) and GmTIP2;5 (right) at 3 Å steps corresponding to the pore shape in (B). Pore axis(X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): pore diameter value in Å.

wilting trait. However, no unique SNPs were identified withincoding sequences in these four slow wilting lines as comparedwith other non-slow wilting lines. The effects of non-synonymousSNP on the pore shape were further analyzed in GmTIP5;1and GmTIP5;2, which have more non-synonymous SNPs in

the protein coding region than other GmTIPs. Interestingly,we find that two non-synonymous SNPs (Gm09_41743311 andGm09_41743354) in GmTIP5;1 do not influence the channel 3Dshape. However, three non-synonymous SNPs (Gm12_895269,Gm12_895302, and Gm12_895476) in GmTIP5;2 have an effect

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FIGURE 4 | Identification and distribution of conserved motifs in soybean TIP protein sequences. Distribution of conserved motifs in soybean TIPmembers. All motifs were identified by Multiple EM for Motif Elicitation (MEME) using the complete amino acid sequences of GmTIP proteins. Different motifs areindicated by different colored boxes numbered 1–10. The annotation of each motif is listed at the bottom. Motif 1 and motif 2 contain the NPA domain.

on the pore shape. Different pore diameter profiles were obtainedbetween Williams 82 genotype and some of the other sequencedgenotypes (Figure 7). For example, the structure of GmTIP5;2in PI 567690 was different than that in Williams 82. These

results indicate that the slow wilting trait in these lines may begoverned by different molecular mechanisms, such as GmTIPexpression level differences or is due to their different 3Dstructures.

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FIGURE 5 | Heatmap of expression profiles of the soybean GmTIPgene family in nine tissues. Relative tissue expression levels of GmTIPsbased on RNA-seq data were used to construct the expression patterns ofsoybean genes. The expression data (Reads Per Kilobase Million) values weremedian-centered and normalized for each gene in different tissue beforetransforming to color scale. The color bar at the bottom shows the range ofexpression values from highest expression level (red) to lowest expressionlevel (green), 0 is the median expression level (Black).

Cloning and Functional Characterizationof GmTIP1;5 and GmTIP2;5 in XenopusOocytesNo non-synonymous SNP was found in GmTIP1;5 and GmTIP2;5in 106 soybean germplasm; however, they were shown to

be differentially expressed in response to drought (GmTIP1;5and GmTIP2;5) and flooding (GmTIP1;5) in different varietiesof soybeans with contrasting phenotypes and were mainlyexpressed in stem where they could be highly important inwater transport during abiotic stress. Further in-depth analysis ofGmTIP1;5 and GmTIP2;5 using X. laevis oocytes was performedto establish a link between differential expression patterns andtrait development. GmTIP2;5 and GmTIP1;5-expressing oocytesshowed a rapid rate of swelling for the first 5 min after beingsubjected to a hypoosmotic solution (Figures 8A–C). After5 min, the rate of swelling in GmTIP2;5 expressing oocytesdeclined as they began to burst. In contrast, GmTIP1;5 expressingoocytes continued to increase in diameter at a rate greater thanthe controls for 20 min. GmTIP2;5-expressing oocytes swelledmore rapidly than GmTIP1;5-expressing oocytes during the first5 min. Mock-injected oocytes displayed a slower rate of swelling,probably due to diffusion of water across the plasma membrane.It was concluded from these results that GmTIP2;5 and GmTIP1;5facilitate the transport of water and are aquaporins.

Some aquaporins transport boron and other solutes inaddition to water (Durbak et al., 2014). To test if theseGmTIPs can transport boron, we applied an isoosmotic solutioncontaining 200 mM boric acid in place of 96 mM NaCl.The GmTIP2;5-expressing oocytes showed significantly increasedswelling with boric acid compared to the control (Figure 8D). Incontrast, control and GmTIP1;5-expressing oocytes did not swell.These results indicated that GmTIP2;5 can transport water as wellas boric acid.

DISCUSSION

GmTIPs Play Important Roles in AbioticStressHere, 23 full-length aquaporin-coding sequences belonging tothe TIP subfamily were identified in the soybean genome. Thenumber of TIPs in the soybean genome is much higher than thoseidentified in species like Arabidopsis, maize, rice, and sorghum(Regon et al., 2014). Several studies have shown differentialexpression of plant aquaporins in response to environmentalstresses in several different species (Alexandersson et al., 2005;Zhu et al., 2005; Ligaba et al., 2011; Cohen et al., 2013; Li et al.,2014). Therefore, it is important to investigate the interactionbetween the expression of GmTIPs and abiotic stress. Thisstudy explored the expression patterns of GmTIP genes insoybean PI 567690 and Pana lines. The soybean genotype PI567690 exhibits significantly lower wilting and less yield lossunder drought condition than the elite cultivar Pana (Pathanet al., 2014). The difference in gene expression between PI567690 and Pana had been investigated by RNA-seq (Princeet al., 2015), and our preliminary data indicated that the PI567690 genotype has a limited transpiration response whenexposed to a high vapor pressure deficit (unpublished data).In the present analysis, we found that the expression level ofseven TIP genes was reduced in slow-wilting soybean linesbut increased in the fast-wilting soybean lines after droughtstress (Figure 6B). At the same time, the expression level of

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FIGURE 6 | Expression profiles of soybean TIP genes under different abiotic stress conditions or in different germplasms. (A) Heatmap representationof expression patterns of soybean TIP genes across the root, leaf, and shoot tissue in Williams 82 under varying water-deficit stress conditions (VMS: very mildstress; MS: mild stress; SS: severe stress; SR: water recovery after severe stress). (B) Expression profiles of the soybean TIP genes in leaves of Pana (fast wilting,drought vs. control), PI 567690 (slow wilting, drought vs. control), PI 408105A (flooding tolerant, flooding vs. control), S99-2281(flooding sensitive, flooding vs.control). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The colorbar at the bottom shows the range of expression values from increased expression (red) to decreased expression (green), 0 means no gene expression patternchanged (Black).

eight different TIP genes was down-regulated or unchangedin the waterlogging tolerant varieties, but upregulated in thewaterlogging susceptible varieties. Only one gene was found tobe up-regulated in slow-wilting lines under drought conditions,and only three genes were found to be up-regulated in thewater-logging tolerant lines. These results are consistent withthe hypothesis that due to lessened water transport in soybeanleaves, the genotype PI 567690 exhibited more resistanceto drought stress. We therefore conclude that GmTIPs mayplay an important role in soybean both in drought andflooding tolerance. However, the gene regulation patterns inroot tissue of water-logging resistant lines should be furtherexplored.

The expression patterns of GmTIPs under a given conditionvaried among different tissues and was complex (Figures 6A,B).

GmTIP1;5 and GmTIP2;5 showed a higher expression in stem,but lower expression in other tissues. Interestingly, GmTIP2;5transcript was up-regulated under varying water-deficit stressin root, but down-regulated after water-recovery. This is theTIP that showed a very rapid water uptake in Xenopus oocytes.GmTIP1;5, which showed a slower, but steady uptake in Xenopusoocytes, was mainly induced under the VMS condition inroot, leaf and shoot, not in other water-deficit conditions.Furthermore, GmTIP1;5 is the only one that showed upregulationin slow wilting lines under drought condition among all TIPgenes. In addition, only one synonymous SNP was located inthe coding sequences of GmTIP1;5 and two synonymous SNPswere located in the coding sequence of GmTIP2;5. Therefore,observed variations in gene expression seem to have prominentroles in functional variation. Further investigation of the water

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FIGURE 7 | Variation in pore morphology of GmTIP5;2 and GmTIP5;2m. GmTIP5;2m contains two amino acid changed (148: A<>G; 159: T<>R) due to twoSNP (Gm12_895269 and Gm_895302). (A) and (B) Cross sections of the proteins showing pore of GmTIP5;2 (A) and GmTIP5;2m (B). (C) and (D) Pore diameterprofile of GmTIP5;2 (C) and GmTIP5;2m (D) at 3 Å steps. Pore axis (X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): porediameter value in Å.

transport function of these two genes in soybean would help toestablish a link between differential expression patterns and traitdevelopment.

To gain further insight into the possible physiologicalfunctions of the members of this large family and aid thedevelopment of genetic engineering in soybean, the TIP thatdirects tissue-specific expression pattern and functions inwater transport would be highly desirable. Overall, a morecomprehensive mechanism will emerge as multiple aquaporintransport functions and integration of the different stress signalsare determined at the whole plant level. Further characterizationof the GmTIP genes involved in abiotic stress resistancegenotypes may aid in developing tolerant germplasm andcultivars.

Water Permeability of GmTIPsAll G. max TIP aquaporins showed the canonical double NPAmotif and a group ar/R SF that are conserved across differentspecies. These results indicate that GmTIPs may play similarroles in regulating water absorption. As such, it is important to

validate representative candidate genes for water transport byusing other methods such as heterologous expression in Xenopusoocytes. Basic GmTIP gene family information and phylogeneticsequence analysis in the present study provide a list of candidategenes that may play important roles in soybean drought andflooding tolerance. The function of two of these GmTIP genes hasbeen demonstrated to be involved in water transport. However,AQPs are not only water transporters but also solute transporters(Maurel et al., 2008). Most AQPs can transport glycerol, urea,boric acid or arsenic as we showed with boron and GmTIP2;5.Yet even though a protein has been predicted to be an aquaporin,it may not transport water. Thus GmTIP candidate genes must befunctionally characterized.

Aquaporins can be further functionally characterized withtransport inhibitors. One such inhibitor, AgNO3 or silversulfadiazine, has been reported to inhibit aquaporin watertransport by binding to cysteine or histidine residues, resulting inblockage of the pore (Daniels et al., 1996; Ishibashi et al., 1997;Niemietz and Tyerman, 2002). Interestingly, the transpirationrate of the slow-wilting cultivar PI416937 is insensitive to silver

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FIGURE 8 | Oocyte Swelling Assays with GmTIP1;5 and GmTIP2;5. The relative diameter of GmTIP-expressing oocytes following exposure to hypoosmotic orboron-containing isosmotic solutions was measured to characterize the osmotic permeability of GmTIP1;5 and GmTIP2;5-containing membranes. (A) Watertransport assay with Xenopus leaevis oocytes in hypoosmotic solution showed that GmTIP2;5-mediated water uptake leads to rapid swelling and bursting (top)compared to the control (bottom). Scale bars = 1 mm. (B) Rate of oocyte swelling in hypoosmotic solution in mock-injected controls (yellow line) vs.GmTIP2;5-expressing oocytes (blue line). Oocytes were observed to burst after 10 min. (C) Rate of oocyte swelling in hypoosmotic solution in mock injected controls(yellow line) vs. GmTIP1;5-expressing oocytes (green line). Results are reported as means ± SEM (N = 5 oocytes). (D) Rate of oocyte swelling in isosmotic boric acidsolution in mock injected controls (yellow line) vs. GmTIP2;5-expressing oocytes (blue line) and GmTIP1;5 (green line). The volume of each oocyte was measured for20 min or until it burst.

treatment, and only a small change in AQP abundance followingsilver treatment was found in this line as compared to sensitivegenotypes (Sadok and Sinclair, 2010b). All these results suggestthat low AQP abundance may underlie the low leaf hydraulicconductivity of PI 416937 and its limited transpiration ratesunder high vapor pressure deficit (Sadok and Sinclair, 2010a,2012; Devi et al., 2015, 2016). The effects of silver ions on watertransport in Xenopus oocytes expressing GmTIP1;5, GmTIP2;5,or other AQPs could help us to identify key candidate geneslinked with the soybean slow-wilting trait. For example, wehypothesize that GmTIP5;1 would be a good target gene forengineering reduced gene expression for improved water stresstolerance, since under severe water-deficit conditions GmTIP5;1was induced in shoot and leaf tissues but showed decreasedexpression in the slow wilting line and flooding tolerant linesunder stress conditions.

Utilization of Natural Variation of GmTIPsAnalysis of whole genome resequencing data provides animmense opportunity to mine natural variants in diversegermplasm (Patil et al., 2016; Valliyodan et al., 2016). Thesoybean germplasm, both wild and cultivated species, providesa wide range of abiotic stress tolerance. This study initiatedcharacterization of GmTIPs by summarizing and analyzing allavailable SNP information, including whether SNPs lead to

synonymous or non-synonymous substitutions, in 106 soybeanlines (Valliyodan et al., 2016). These 106 soybean lines representdiverse lines (wild types, landraces and elite lines), includingfour slow wilting soybean lines (PI 416937, PI 471938, PI567690, and PI 567731). Aside from the expression level, geneticvariation (natural or induced) also may impact the functionalityof aquaporin genes and cause a variation in phenotypes. To ourknowledge, this is the first report of natural variation in soybeanfor TIP genes. With this information, the correlation between theSNP haplotype and the phenotype (slow wilting score) for these106 lines can be calculated to further characterize the associationbetween the GmTIP genetic background and the slow-wiltingtrait. Furthermore, the association analysis can be extended to thewhole MIP gene family and the gene promoter region to betterunderstand the mechanism of slow wilting at the genomic level.

It has been reported that plant plasma membrane aquaporinsare deactivated by dephosphorylation under conditions ofdrought stress, or by protonation of a conserved histidine residuefollowing a drop in cytoplasmic pH due to anoxia during flooding(Tournaire-Roux et al., 2003; Törnroth-Horsefield et al., 2006).Analyzing the detailed effects of all non-synonymous SNPs on theexisting crystal structure of plant AQPs (for examples: AtTIP2;1,SoPIP2;1) (Törnroth-Horsefield et al., 2006) in some interestingsoybean lines may enable us to better understand the structurechanges and mechanisms of water transport regulation.

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CONCLUSION

In this study, the soybean TIP gene family was identified based onthe new soybean genome annotation. Twenty-three TIP memberswere assigned to five subfamilies based on sequence similarity andphylogenetic relationship. The modulation of expression profilesof these 23 GmTIP genes was examined with deep transcriptomesequencing under drought or flooding conditions and duringplant development. The potential roles of these genes intransport of substrates were also discussed. The identified naturalvariations in this gene family from 106 soybean germplasmswill benefit future gene functional analysis and utilization. Wealso demonstrated that GmTIP1;5 and GmTIP2;5 can function aswater transporters in oocytes. All results presented here representan important resource for designing experiments for functionalvalidation of candidate genes in plant development and abioticstress responses, and for developing soybean germplasm withimproved abiotic stress tolerance.

AUTHOR CONTRIBUTIONS

LS and RD designed the experiments, analyzed data, andprepared the manuscript. NN, SMP, LS and WG worked onthe oocytes experiments. GP, RD, and BV analyzed the natural

variation SNP data. RD and LS worked on defining pore-liningresidues and solute prediction. SJP and RM were involved in thegene expression pattern analysis. HN conceived and supervisedthe project. All authors have read, revised, and approved themanuscript.

FUNDING

This project was funded by the Missouri Soybean MerchandisingCouncil and United Soybean Board.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Amanda Durbak and Dr.Paula McSteen, University of Missouri, for help with oocyteswelling assays. We thank Theresa Musket and Kevin Ngo forproof-reading and editing the manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found onlineat: http://journal.frontiersin.org/article/10.3389/fpls.2016.01564

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Song et al. Soybean TIP Gene Family Analysis

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2016 Song, Nguyen, Deshmukh, Patil, Prince, Valliyodan, Mutava, Pike,Gassmann and Nguyen. This is an open-access article distributed under the termsof the Creative Commons Attribution License (CC BY). The use, distribution orreproduction in other forums is permitted, provided the original author(s) or licensorare credited and that the original publication in this journal is cited, in accordancewith accepted academic practice. No use, distribution or reproduction is permittedwhich does not comply with these terms.

Frontiers in Plant Science | www.frontiersin.org 15 October 2016 | Volume 7 | Article 1564